This research will focus on molecular labeling of secretory vesicles and cytoskeletal elements involved in the secretion of adhesive material and wall components to the surface of fungal cysts and hyphal tips. The study should provide-a clearer understanding of the role of an assortment of vacuoles, vesicles and cisternae which develop at specific times in the life cycle of these fungi and a clearer picture of the role which microfilaments and microtubules play in guiding these vesicles to sites of secretion. Specifically, the developmental pathway of a prominent secretory vesicle, the "peripheral vesicle", will be followed from synthesis to secretion. Gold-labeled lectins will be used to label thin sections prepared for transmission electron microscopy to trace the development of the vesicles in germinating cysts and oospores. Neither of these synthesized upon germination. Samples of germinating spores will be fixed at selected intervals for transmission electron microscopy, stained with gold labeled lectins, and examined for the involvement of the Golgi apparatus, endoplasmic reticulum, or other organelles in the synthesis of these vesicles. As the peripheral vesicles migrate to the periphery of zoospores of these fungi, the exocytosis of adhesive material to the cell surface or the internalization of these vesicles will again be monitored with gold labeled lectins and transmission electron microscopy. Finally, rhodamine conjugated phalloidin and fluorescein-labeled antibodies will be used to visualize microfilaments and microtubules of the cytoskeleton, respectively. Fluorescence microscopy will show the involvement of the cytoskeleton in directed cellular secretion and may tie in with the role of calcium in stimulating encystment and hyphal tip growth in these fungi. This project will provide participating students with hands-on experience with modern molecular techniques for localizing cellular components and cytoskeletal elements.